ProjectSummary Rapidlyproliferatingcancercellsgeneratereactiveoxygenspecies(ROS)thatifleftuncheckedinhibitcell growth.Tocounterthisstress,cancercellsandinparticularnonsmallcelllungcancers(NSCLC)relyonthe activationoftheNRF2transcriptionfactor,leadingtothemassiveupregulationofkeymetabolicand detoxificationproteinsneededtorestoreredoxbalance.WhiledirectlytargetingNRF2withchemicalinhibitors ischallenging,wehypothesizedthathyperactivationofthispathwaywouldleadtoanalterationofspecific signalingandmetabolicpathwaysrequiredfortheproliferationofthesecells(co-dependencies),which themselvescouldbeinhibitedwithsmallmolecules.Toidentifytheseco-dependenciesinNSCLC,weenriched forproteinscontainingreactivecysteines,whichcanbeusedasachemicalhandletodevelopinhibitors.This chemicalproteomicsscreenidentifiedhundredsofreactivecysteinesregulatedbyNRF2,includingacryptic cysteine(C274)intheorphanreceptorNR0B1.NR0B1expressionisseverelyrestrictedtothoseNCSLCcells andpatienttumorswithderegulatedNRF2signaling,whereitfunctionsaspartofmultimerictranscriptional complextosupporttheNRF2geneexpressionprogram.AsC274inNR0B1isnecessaryforNR0B1-complex formation,weexploitedthisresiduetodevelopasmallmoleculeinhibitorthatcovalentlybindstoit, subsequentlydisruptingtheprotein-proteininteractionsofNR0B1andblockingthegrowthofNRF2-dependent cells,butnotNRF2-independentcells.Thus,wehaverevealedNR0B1asadruggableco-dependencyofthe NRF2pathway.Inthisgrantapplication,webuildonourresearchonNR0B1andfurtheridentifyco-dependent pathwayswithNRF2thatcanbepharmacologicallyinterrogated.Usingapowerfulchemoproteomic framework,wewillcomprehensivelydefineNRF2co-dependenciesby:1)mappingthelandscapeofcysteine reactivityregulatedbyNRF2inlungxenograftmodels,allowingustoidentifycysteinesonkeyproteinsinthe NRF2pathway,whichmaybecometargetableopportunitiesinvivo2)undertakingasmallmoleculescreento identifycompoundsthatselectivelyinhibittheproliferationofNRF2-dependentNSCLCcelllines.Importantly, integratingachemoproteomicplatformintothisscreen,willallowfortherapididentificationofco-dependent proteins,offeringanunparalleledmapofdruggableNRF2co-dependencies.Theresearchproposedherein, takesfulladvantageofadvancedcancermodelsandchemoproteomictechnologiestoreveal pharmacologicallytractableproteinswhichareneededfortheproliferationofNRF2-addictedcellsandmay provideageneralizableplatformforinhibitinggeneticallydefinedcancers.Thesestudieswillnotonlyprovidea comprehensiveunderstandingofNRF2biologybutmightalsolaythefoundationfortranslationaltherapeutics benefitinglungcancerpatientswithderegulatedNRF2signaling.